University of Massachusetts Amherst ScholarWorks@UMass Amherst Masters Theses 1911 - February 2014 1985 The biology and ecology of the pinewood nematode, Bursaphelenchus xyophilus (Steiner and Buhrer) Nickle, in Massachusetts / Anne E. Dorrance University of Massachusetts Amherst Follow this and additional works at: https://scholarworks.umass.edu/theses Dorrance, Anne E., "The biology and ecology of the pinewood nematode, Bursaphelenchus xyophilus (Steiner and Buhrer) Nickle, in Massachusetts /" (1985). Masters Theses 1911 - February 2014. 3388. Retrieved from https://scholarworks.umass.edu/theses/3388 This thesis is brought to you for free and open access by ScholarWorks@UMass Amherst. It has been accepted for inclusion in Masters Theses 1911 - February 2014 by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. THE BIOLOGY AND ECOLOGY OF THE PINEWOOD NEMATODE BURSAPHELENCHUS XYLOPHILUS (STEINER AND BUHRER) NICKLE, IN MASSACHUSETTS A Thesis Presented By ANNE ELIZABETH DORRANCE Approved as to style and content by: j'' v f-- * (, o. -V--- of Committee ■Dr. Terfy^L Tattar, M^ber Dr. Joseph S. Elkinton, Member / ..4 /' / z / , i i ' 1 i / Dr. T. Michiel Peters, Member Dr. Mark S. Mount, Department Head Department of Plant Pathology THE BIOLOGY AND ECOLOGY OF THE PINEWOOD NEMATODE BURSAPHELENCHUS XYLOPHILUS (STEINER AND BUHRER) NICKLE, IN MASSACHUSETTS A Thesis Presented By ANNE ELIZABETH DORRANCE Submitted to the Graduate School of the University of Massachusetts in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE February 1985 Department of Plant Pathology To Mom, Dad, Laurie and Jean ACKNOWLEDGEMENTS My sincere thanks are extended to my major advisor, Dr. Richard A. Rohde, for his advice and direction over the course of this project. I would also like to thank the other members of the committee. Dr. Tattar, Dr. Elkinton and Dr. Peters, for their comments throughout the course of this study. My sincere thanks are also extended to Mr. Dick Kelliher, Mr. Charles Burnham, Mr. Ed Budnick, Mr. Fred Haywood of the Bureau of Insect and Pest Management and Bill Wilcox of Extension for their help with the state survey. I gratefully acknowledge the aide of Mr. Daniel Plourde, Mr. Kevin Carr, Mr. Anthony Medeiras, at the Department of Plant Pathology, University of Massachusetts, as well as Mr. Brian Dale, Mr. Steve Kinsman and Mr. Doug Baptiste of the Public Works Department, Town of Falmouth, for their help in harvesting all my plots. I would also like to acknowledge Meg Worcester for her patience and typing exper¬ tise. A very special thanks to my husband, Tom, without whom most of this could not have been completed. IV TABLE OF CONTENTS Chapter I. INTRODUCTION . 1 II. LITERATURE REVIEW . 6 Disease Cycle . 6 The Nematode.7 Insect Associates . 13 Histopathology of Infected Pines . 18 Other Factors Associated with Pine Wilt Disease ... 19 III. MATERIALS AND METHODS.24 Inoculum Preparation . 24 Seedling Inoculations . 25 Plantation Inoculations . 26 State Survey.29 Histopathology . 29 Vector Studies . 30 IV. RESULTS AND DISCUSSION.32 Seedling Studies . 32 Field Studies. State Survey.89 Vector Studies . 82 V. LITERATURE CITED . 85 V LIST OF TABLES 1. Insects associated with Bursaphelenchus xylophilus in Japan and the United states. 2. Summary of field inoculations of Red (1,3,4), Japanese black (2) and Pitch pine with xylophi1 us in Massachusetts .... 27 3. Number of three-year-old pine seedlings with Pine Wilt Symp¬ toms after inoculation with three different populations of B^. xylophilus and Botrytis spores, 24 days after inoculation Tn the greenhouse 1983 . 4. Comparison of Japanese black, white and Austrian pine three- year-old seedlings in greenhouse studies at 60, 54 and 59 days, respectively, after inoculation . 35 5. Recovery of xylophi1 us (FTF-84) from Japanese black pine (Pinus thunbergiana), 105 days after inoculation . 40 6. Field inoculations of Larch (Larix laricina)at Montague fields, 1984 . 7. Pathogenicity of Bursaphelenchus xylophilus to branches of red pine trees . .. 8. B. xylophilus populations in the stem inoculated red pine X^Pinus resinosa) trees in July, 1984, at various locations in Massachusetts .. 9. Comparison between girdled and nongirdled B^. xylophilus stem inoculated red pine at Mt. Toby, Massachusetts, 1984 . 58 10. Cerambycids collected from various sources of B^. xylophilus infested logs, 1983 and 1984 .63 vi LIST OF ILLUSTRATIONS Red pine (Pinus resinosa) at the University of Massachusetts at Amherst exhibiting reddish brown needle coloration similar to pine wilt disease. ^ Bursaphelenchus xylophilus female with overlapping vulval flap. 3. Bursaphelenchus xylophilus male with rosethorn shaped spicules with an expanded tip. 4. Scots pine (Pinus sylvestris L.) inoculated with 2,000 ]B. xylophilus from Missouri, Massachusetts and Botrytis spores . 34 5. The number of nematodes/gram of fresh weight of wood oyer time in three-year-old white, Japanese black and Austrian pine seedlings.. Larch (Larix laricina) seedling developing symptoms of pine wilt following inoculation with xylophilus . 43 Japanese black pine (Pinus thunbergiana) in March, 1984, with stunted buds and slight yellow discoloration of needles com¬ pared with control. 8. Resin soaking in B^. cinerea and xylophilus inoculation sites in red pines from Mt. Toby, September, 1984 . 51 9. Inoculation wound from xylophi1 us inoculated red pine at Mt. Toby, September, 1984 . 10 Bursaphelenchus xylophi 1^ stained with Goodey's formula from inoculation site of a red pine. Tree was inoculated with 30,000 nematodes in July, 1983, and harvested September, 1984 . 11. Tangential section of red pine following inoculation with B. xylophilus. Fusiform ray with destroyed epithelial cells . 55 12. Tangential section of red pine with healthy fusiform ray fol¬ lowing inoculation with Botrytis . 55 13. Locations of pinewood nematode, Bursaphelenchus xylophilus from declining pines in Massachusetts, sites of declining pines or larches where no pinewood nematodes were recovered . 61 CHAPTER I INTRODUCTION The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer 1934) Nickle 1970, formerly B. liqnicolus Mamiya and Kiyohara 1969, was first recovered in Massachusetts from the sapwood of a dying red pine (Pinus resinosa Ait.) on Cape Cod (Rohde 1983). This plant parasitic nematode had been reported to cause a rapid wilt disease of most Pinus spp. trees in Japan known as pine wilt (Mamiya 1972a). The first noticeable symptom of disease is a change in needle coloration. Infected trees change from dark green to grey- green, then to yellow green, followed by the bright reddish brown of dead trees (Figure 1). In warmer climates of Japan this change may occur in only six to eight weeks, while in cooler climates, trees which become infected in the summer may not die until the following spring (Mamiya 1983). Another important symptom of pine wilt is a decrease in the oleoresin flow from bark wounds or from broken branches (Mamiya 1972). Whole trees may be affected or pine wilt disease may be limited to one or several diseased branches of a single tree (Dropkin ^ al_. 1981, Malek and McClary 1981, Malek ^ ai. 1982 and Malek and Appleby 1984). At various times pine wilt was reported to be caused by many different agents, such as bark engravers, drought, off-site planting, fungal infection, adverse soil conditions, rodent injury, lightning. 1 2 Figure 1. Red pine (Pinus resinosa Ait.) at the University of Massachusetts at Amherst exhibiting reddish brown needle coloration similar to pine wilt disease. 4 and other forms of stress (Kobayashi 1984, Malek ^ ll- 1982 and Robbins 1979). In 1969, Kiyohara and Tokishige first indicated the nematode, B_. xylophilus, as the primary pathogen of pine wilt disease in Japan. There was at the time of discovery only one other known instance of a migratory endoparasitic nematode causing a vascular disease. Rhadinaphelenchus cocophilus, which causes red ring of coconut, is an economically important disease of coconut and oil palms in the West Indies and South America (Blair and Darling 1968). The only other known wilt disease of pine had been reported by Basham (1970) on loblolly pine and was caused by a blue-stain fungus Cera- tocystis (Mamiya 1983). Pine wilt disease first occurred in 1905 in Japan and has since developed into a serious epidemic there. It has been estimated that twenty-five percent of Japan's 2.6 million ha of pine forest has been affected. Losses of pine 1.5 million m^ was lost. In 1980, the Japa¬ nese government spent 35 million dollars on control of pine wilt (Nickle ^ 1981, Mamiya 1980, 1983a,b). Since the pinewood nematode's discovery in the United States in 1979 by Dropkin and Foudin, much interest, research, and controversy has surrounded pine wilt disease. There have been several symposia, a joint meeting between U.S. and Japanese scientists and approximately 50 papers published by American researchers. In some areas of the United States the pinewood nematode has proven to be of economic importance (Adams and Morehart 1981, 1982, Bergdahl 1982, Birchfield et al. 1981, Dropkin 1982, Dropkin ^ 1981, Nickle 1982, 5 Robbins 1982, Wingfield 1982, and Wingfield & Blanchette 1984). In Illinois, losses of trees in Christmas tree plantations and in urban settings due to pine wilt disease have been reported (Malek and Appleby 1984 and Malek ^ 1981). In Massachusetts, i. xylophilus has been isolated from dying Japanese black (Pinus thunbergiana Franco), red (K resinosa Ait.) and Scots pine (^. sylvestris L.) on Cape Cod (Dorrance and Rohde, unpublished). Japanese black pines originally were planted in 1895 in Wauwinet, Nantucket, because of their salt tolerance. They are commonly used to help prevent soil erosion and to stabilize beaches (Jones 1930). These trees have also been used as landscape trees for many homes. Since 1983, losses of Japanese black pines due to pine wilt disease have been reported along the eastern shore line from Cape Cod to N. Carolina (Nickle 1984). Death of Japanese black pines on Cape Cod has also been associated with blue-stain fungi and black turpentine beetles (Highley 198^).